Post-translational modification of proteins is an important mechanism required for many cellular functions, including the mediation of protein-protein interactions, enzymatic activity, degradation, localization of proteins to cellular compartments, and maintenance of stability. The modifications usually occur via specific enzymatic reactions that catalyze the transfer of various chemical/molecular groups to specific amino-acid residues of target proteins. Some well known examples of post-translational modification include phosphorylation, acetylation, methylation, glycosylation, and ubiquitination. The unique feature of ubiquitination is that the moiety that is transferred to the target protein is itself a polypeptide (ubiquitin). Ubiquitination usually serves as a signal in the cell to target-modified proteins for degradation by the 26S proteosome. The molecular mechanism of ubiquitin conjugation and its regulation have been the subject of extensive studies.
A number of proteins have been discovered that share sequence similarities with ubiquitin. An early example of these proteins is “SUMO” (“small ubiquitin-like modifier”). Since its discovery several years ago, the study of SUMO-modification has evolved into a very important and productive field. Efforts have been dedicated to characterizing the enzymology of the SUMO-modification pathway and identifying target proteins for SUMO. These studies have revealed the following important features of the SUMO protein family. SUMO and the SUMO-modification pathway are highly conserved across the eukaryotes (Müller at al., “SUMO, Ubiquitin's Mysterious Cousin,” Nature Reviews/Molecular Cell Biology 2:202–210 (2001)). While invertebrates have a single SUMO gene, the SUMO family in vertebrates consists of three genes: SUMO-1, -2, and -3. SUMO-1 (also known as Sentrin, UBL1, PIC1, and GMP1), is the prototype member of the ubiquitin-like family of protein modifiers and was isolated by several labs independently (Boddy et al., “PIC 1, A Novel Ubiquitin-Like Protein Which Interacts with the PML Component of a Multiprotein Complex that is Disrupted in Acute Promyelocytic Leukaemia,” Oncogene 13(5):971–982 (1996); Mahajan et al., “A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2,” Cell 88:97–107 (1997); Matunis et al., “A Novel Ubiquitin-like Modification Modulates the Partitioning of the Ran-GTPase-activating Protein RanGAP1 Between the Cytosol and the Nuclear Pore Complex,” J. Cell Biol. 135:1457–1470 (1996); Matunis et al., “SUMO-1 Modification and its Role in Targeting the RanGTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex, “J. Cell Biol. 140:499–509 (1998); Okura et al., “Protection Against Fas/APO-1-and Tumor Necrosis Factor-Mediated Cell Death by a Novel Protein, Sentrin,” J. Immunol. 157(10):4277–4281 (1996); Shen et al., “UBL1, A Human Ubiquitin-Like Protein Associating with Human RAD51/RAD52 Proteins,” Genomics 36(2):271–279 (1996)). Conjugation by SUMO-1 has received most of the research efforts, while relatively little is known about the modifications by SUMO-2 and SUMO-3. The enzymes for SUMO-modification are distinct from those involved in ubiquitination, but the overall enzymology of the SUMO-modification pathway appears to parallel that of the ubiquitin-conjugating pathway and involves a SUMO-activating enzyme (“E1”), a SUMO-conjugating enzyme (“E2”), and a ligase activity (“E3”). Examples of E3 activity have only been discovered very recently (Johnson and Gupta, “An E3-like Factor that Promotes SUMO Conjugation to the Yeast Septins,” Cell 106: 735–744 (2001); Takahashi et al., “A Novel Factor Required for the SUMO1/Smt3 Conjugation of Yeast Septins,” Gene 275:195–315 (2001); Kahyo et al.,” Involvement of PIAS1 in the SUMO Modification of Tumor Suppressor p53,” Molecular Cell 6: 713–718 (2001)). In contrast to ubiquitination, SUMO-modification generally does not promote the degradation of the target proteins. Instead, it appears to play important roles in modulating target protein function(s). Identified targets for SUMO-modification include proteins that play important roles in various aspects of cell function, such as tumor suppressors p53 and PML, the nuclear-pore component RanGAP1, the proto-oncogene Mdm-2, and the NF-kB regulator IkB.
SUMO-1 shows only an 18% homology to ubiquitin. SUMO-attachment to a protein substrate is reversible and usually does not result in SUMO-chain formation. Also in contrast to ubiquitination, which targets proteins for degradation, sumolation seems to enhance the stability of proteins and/or modulate specific protein-protein interactions. In addition, SUMO conjugation can also result in specific trafficking and localization of target proteins.
In an effort to identify proteins involved in double strand break repair of DNA, Shen et al., showed that SUMO-1 interacts with RAD51/RAD52, a protein complex formed during DNA repair and recombination (Shen et al., “UBL1, A Human Ubiquitin-Like Protein Associating with Human RAD51/RAD52 Proteins,” Genomics 36(2):271–279 (1996)).
Other studies isolated SUMO-1 as a factor which binds to the ‘death domain’ of the Fas/APO-1 and the TNFR1 receptors and therefore, plays a role in apoptosis. These studies showed that when overexpressed, SUMO-1 provided protection against both Fas/APO-1 and TNF-induced cell death (Okura et al., “Protection Against Fas/APO-1-and Tumor Necrosis Factor-Mediated Cell Death by a Novel Protein, Sentrin,” J. Immunol. 157(10):4277–4281 (1996)). Northern blot analysis of SUMO-1 showed expression in all tissues, with the highest levels being in the heart, skeletal muscle, testis, ovary, and thymus.
SUMO-1 was also shown to be involved in nuclear protein import by conjugating to the 70 kD nuclear pore protein RanGAP1, which could then interact with RanBP2, resulting in a complex which is necessary for nuclear protein import (Mahajan et al., “A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2,” Cell 88:97107 (1997); Matunis et al., “SUMO-1 Modification and its Role in Targeting the RanGTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex,” J. Cell Biol. 140:499–509 (1998)). It has been shown that SUMO-1 conjugation is carried out by Ubc9, an enzyme equivalent to the E2 enzyme of ubiquitin conjugating pathways (Gong et al., “Preferential Interaction of Sentrin with a Ubiquitin-Conjugating Enzyme, Ubc9,” J. Biol. Chem. 272(45):28198–28201 (1997); Johnson and Blobel, “Ubc9p is the Conjugating Enzyme for the Ubiquitin-Like Protein Smt3p,” J. Biol. Chem. 272:26799–26802 (1997); Lee et al., “Modification of Ran GTPase-activating Protein by the Small Ubiquitin-Related Modifier SUMO-1 Requires Ubc9, an E2-type Ubiquitin-Conjugating Enzyme Homologue,” J. Biol. Chem. 273:6503–6507 (1998); Saitoh et al., “Ubc9p and the Conjugation of SUMO-1 to RanGAP1 and RanBP2,” Curr. Biol. 8:121–124 (1998); Schwarz et al., “The Ubiquitin-Like Proteins SMT3 and SUMO-1 are Conjugated by the UBC9 E2 Enzyme,” Proc. Natl. Acad. Sci. USA 95(2):560–564) (1998)). SUMO-1 interaction and modification have also been documented for the tumor suppressor protein PML and its nuclear body partner Sp100 (Boddy et al., “PIC 1, A Novel Ubiquitin-Like Protein Which Interacts with the PML Component of a Multiprotein Complex that is Disrupted in Acute Promyelocytic Leukaemia,” Oncogene 13(5):971–982 (1996); Stemsdorf et al., “Evidence for Covalent Modification of the Nuclear Dot-Associated Proteins PML and Sp100 by PIC1/SUMO-1,” J. Cell Biol. 139(7):1621–1634 (1997)). Other examples of targets of SUMO modification include the tumor suppressor p53, the proto-oncogene Mdm2, and the NF-kappaB regulator I-kappaB (Müller at al., “SUMO, Ubiquitin's Mysterious Cousin,” Nature Reviews/Molecular Cell Biology 2:202–210 (2001)).
Those protein modifiers which are called ‘ubiquitin-like’ modifiers (“UBLs”), of which SUMO is a prime example, function as modifiers in a manner analogous to ubiquitin, i.e., the modifier protein is conjugated to the protein it is modifying. Other examples of UBLs include NEDD8 and Apgl2 (Müller at al., “SUMO, Ubiquitin's Mysterious Cousin,” Nature Reviews/Molecular Cell Biology 2:202–210 (2001)). A second group of proteins, designated ‘ubiquitin-domain proteins’ (“UDPs”), have been identified containing domains that are related in sequence to ubiquitin. In contrast to UBLs, UDPs do not conjugate to other proteins (Müller at al., “SUMO, Ubiquitin's Mysterious Cousin,” Nature Reviews/Molecular Cell Biology 2:202–210 (2001)).
Given the importance of SUMO-modification (referred to here as sumolation), identification of additional SUMO targets are certainly of great research interest, which should not only reveal more aspects of cellular life regulated by SUMO-modification, but may also provide novel clues for developing therapeutic drugs that intervene or regulate these important cellular processes. Currently, there are only two main approaches for detecting SUMO-modification. The in vitro assay attempts to reconstitute the modification reaction using purified or partially purified components (either from cell extracts or from a recombinant source) such as GST-SUMO, GST-UBC9 (the E2 enzyme), and the E1 enzyme. The protein to be tested is usually in a radio-labeled form (e.g., produced from in vitro translation) or in a purified recombinant form. If the test protein undergoes sumolation, the modified form migrates more slowly than the apo-form in SDS-PAGE, which can be detected by either Western blot or autoradiography. However, this in vitro approach is generally inefficient, either due to the intrinsic property of the modification system, or, more likely, due to the lack of E3 activity in the in vitro reactions. The potential problem of lack of E3 activity may not be easily solved, despite the recent identification of the first examples of E3 activities for the SUMO-conjugation pathway. The ubiquitination pathway employs a large number of distinct E3 enzymes, which is understandable, given the fact that substrate proteins are diverse, and that E3 plays an important role in the recognition of substrate specificity. Thus, it is likely that many E3s for the SUMO-pathway remain to be discovered.
The second current approach attempts to directly detect SUMO-modification of candidate proteins in cells. Usually, the candidate protein (either expressed endogenously or by transfection) is immunoprecipitated from cell lysates by an appropriate antibody (“Ab”), resolved on an SDS-PAGE, and then Western-blotted using an appropriate Ab against this protein and/or against SUMO. The advantage of this in vivo system is that the modification reaction occurs in the cell and utilizes the cellular enzymatic system. Nevertheless, there are several potential drawbacks to this approach: 1) the feasibility to carry out this approach is limited by the availability of appropriate Abs that exhibit desired specificity and sensitivity; 2) the isopeptide bond of sumolation is very sensitive to protease attack and can be rapidly lost during cell lysis; 3) the relatively harsh conditions that are used to lyse the cells in order to inactive these proteases can further limit the utilization of appropriate Abs to carry out the analysis; and 4) currently published studies usually show that even though a candidate protein is SUMO-modified in cells, the detectable sumolated form is only a very small portion of the total candidate protein. Therefore, the sensitivity of the detection is also an issue in existing methods.
While much is known about SUMO and other factors involved in post-translational modification of cellular proteins, there are currently no dependable methods of identifying proteins which are post-translational modifiers (termed here as “post-translational modifier polypeptides”, or “PMPs”), regulators of PMPs, or target proteins of PMPs. The present invention seeks to overcome these and other deficiencies in the art.